The Hox complex of genes affects both development and disease, but the pathways by which Hox transcription factors regulate downstream target genes remain largely undefined. Targeted gene disruption studies have confirmed that these genes affect anteroposterior patterning processes, but phenotypes of single mutant animals are generally mild, pleiotropic, demonstrate incomplete penetrance and have variable expressivities. Because of the nature of these defects, very little is known regarding downstream targets of Hox genes or the molecular pathways in which Hox genes operate. By removing all six functional copies of the Hoxll paralogous genes, we generated animals that have no kidneys. The metanephric blastema forms early in development, but no ureteric bud induction occurs. This phenotype occurs with 100% penetrance. By examining this phenotype molecularly, we have been able to identify factors downstream of the Hoxll paralogous genes. Six2 a member of the conserved Pax-Eya-Six pathway, and Gdnf, the inducing ligand responsible for signaling to the Wolffian duct to initiate ureteric bud induction, are not expressed in the Hoxll paralogous mutants. Our preliminary data indicate that Six2 is a direct downstream target of the Hoxll paralogs, and that Hoxll paralogs directly interact with Eyal and Pax2 to regulate Six2 expression. As each of these genes has also been implicated in the regulation of Gdnf, the inducing ligand, defining their interactions is critical to understanding the molecular basis of kidney development. Finally, complimentary new findings indicate that HoxlO paralogous genes also play a critical role in nephrogenic development by as yet undefined mechanisms. In our view, this system is ideal for identifying downstream targets of Hox genes and factors with which they regulate expression. The paralogous nature of the Hox genes make it very likely that these genes act as modifiers of disease proccesses in conjunction with other factors in their regulatory network. Mutations in Pax, Eya and Six genes have been demonstrated in human cases of Branchio-Oto-Renal Syndrome as well as Renal- Coloboma Syndrome. It is thus very likely that Hox genes influence the severity of these syndromes in humans. Using the kidney as a model organ system, we will examine the interaction between these highly conserved groups of developmental regulators and define the previously undescribed molecular relationships between Hox genes and the Pax-Eya-Six/Gdnf regulatory pathway in nephrogenesis. We hypothesize that a conserved interaction of Hox genes with the Pax-Eya-Six regulatory network specifies ureteric bud induction and patterns the developing kidney. Relevance: These studies will provide key insights into the molecular and mechanistic basis of Hox regulation in kidney development and disease.